WO1991013164A1 - Procede biochimique de production d'oligosaccharides - Google Patents

Procede biochimique de production d'oligosaccharides Download PDF

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Publication number
WO1991013164A1
WO1991013164A1 PCT/SE1991/000165 SE9100165W WO9113164A1 WO 1991013164 A1 WO1991013164 A1 WO 1991013164A1 SE 9100165 W SE9100165 W SE 9100165W WO 9113164 A1 WO9113164 A1 WO 9113164A1
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WO
WIPO (PCT)
Prior art keywords
ome
reaction
acceptor
acceptor substance
oligosaccharide
Prior art date
Application number
PCT/SE1991/000165
Other languages
English (en)
Inventor
Kurt Nilsson
Original Assignee
Procur Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Procur Ab filed Critical Procur Ab
Priority to EP91905105A priority Critical patent/EP0517766B1/fr
Priority to DE69113028T priority patent/DE69113028T2/de
Publication of WO1991013164A1 publication Critical patent/WO1991013164A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • - carbohydrates are antigenic determinants (for example blood group antigens) ;
  • glycosidases Both hydrolases (glycosidases, EC 3.2) and glycosyl- transferases (EC 2.4) can be used for synthesis (glycosidases: see Nisizawa et al, in "The Carbohydrates, Chemistry and Biochemistry", 2nd ed. , vol IIA, pp. 242-290, Academic Press, New York, 1970) .
  • glycosidases reversed hydrolysis (equilibrium reaction) or transglycosylation (kinetic reaction) are often used to obtain synthesis (see e.g. K.G.I. Nilsson, Carbohydr. Res., vol. 167, pp. 95-103, 1987, Trends in Biotechnology., vol. 6, pp.
  • glycosidases are abundant and can often be used directly without purification. However, disadvantages with glycosidases are that relatively low yields and wrong product isomers often are obtained. Yields of disaccharides of between 5 and 45 % have been obtained in transglycosylation reactions with glycosidases.
  • the method according to the present invention allows glycosidase-catalysed synthesis of glycoconjugate oligosaccharide compounds in considerably higher yields than achieved with previous methods.
  • This is achieved according to the invention by using a high concentration of dissolved acceptor substance in transglycosylation reactions so that the reaction mixture (usually solvent, glycosyl donor, glycosyl acceptor, enzyme and buffer salts) is saturated to 30 % or more with the acceptor substance at other conditions of the reaction.
  • the high concentration of acceptor is achieved by for example dissolving more and more of the acceptor substance at a temperature which is close to the boiling point of the solvent (usually buffered water).
  • very high acceptor concentrations be used (for example ca 1 M concentration or higher of nitrophenyl glycosides and 2 M or higher of methyl glycosides) , which are stable (the acceptor does not precipitate, crystallise from the solution) at the temperature which is used for the transglycosylation reaction, and which are substantially higher than what have been used in previous transglycosylation methods (e.g. 0.15 M nitrophenyl glycoside and 0.6 M of methyl glycoside) for the synthesis of glycoconjugate oligosaccharide structures.
  • the yield of transglycosylation products calculated on donor glycoside cf. the reaction scheme for transglycosylation reactions in the articles referred to on p.
  • disaccharides and higher oligosaccharides can also be dissolved in considerably higher concentrations than what have been used in previous transglycosylation reactions and thus, according to the invention exoglycosidases can be used for preparative synthesis of tri-, tetra- and higher oligosaccharides in satisfactory yields.
  • the aglycone can be a glycosidically bound flourine or an 0-, N-, C-, or S-glycosidically bound ( C - or ⁇ -configuration) aliphatic or aromatic compound (as for example methyl, ethyl, 2-bromoethyl, (CH j ) COOMe, n>l, allyl, benzyl, trimethylsilylethyl, amino acid, a derivative thereof, peptide, a derivative thereof, nitrophenyl, etc) .
  • aliphatic or aromatic compound as for example methyl, ethyl, 2-bromoethyl, (CH j ) COOMe, n>l, allyl, benzyl, trimethylsilylethyl, amino acid, a derivative thereof, peptide, a derivative thereof, nitrophenyl, etc
  • glycosyl donors may be mentioned (X- or ⁇ -bound nitrophenyl-, methyl- or allyl-glycosides of the monosaccharides above, lactose, dimannose and raffinose. Oligosaccharides and glycosides of disaccharides or oligosaccharides which are substrates for endoglycosidases are also suitable as donor substrates.
  • the concentration of the glycosyl donor in the reaction mixture is selected with regard to the oligosaccharide which is to be synthesised and also with regard to the properties of the enzyme and therefore do not restrict the use of the invention.
  • addition of the donor in smaller portions may be advantageous in order to minimise the risk that the donor also acts as an acceptor (unless this is desired; see the example below with GalD-OPhNO* as donor) .
  • glycosidase which is employed to catalyse the transglycosylation reaction
  • another type of enzyme can be added to the reaction mixture to degrade or convert the hydrolysis product of the donor substance (for example, mannose is formed when a mannose glycoside is used as donor) .
  • mannose is formed when a mannose glycoside is used as donor
  • an enzyme that selectively converts the hydrolysis product to a product which inhibit the glycosidase-reaction to a minimal extent is preferentially used.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

Un procédé permet de synthétiser des composés oligosaccharides composés ou constitués d'un fragment ou d'un analogue du composant glucidique d'un glycoconjugué. On effectue la synthèse à partir d'un donneur et d'un accepteur en utilisant au moins une glycosidase, dans une réaction de transglycosylation, et en utilisant une concentration très élevée d'accepteur dissous, de sorte que le mélange de réaction est saturé à plus de 30 % avec l'accepteur dans d'autres conditions de réaction. On obtient cette concentration élevée d'accepteur en dissolvant par exemple de plus en plus d'accepteur à proximité du point d'ébullition du solvant. Ce procédé permet d'obtenir par des réactions de transglycosylation catalysées par glycosidase de grandes quantités du produit de transglycosylation avec le glycoside donneur.
PCT/SE1991/000165 1990-03-02 1991-03-01 Procede biochimique de production d'oligosaccharides WO1991013164A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP91905105A EP0517766B1 (fr) 1990-03-02 1991-03-01 Procede biochimique de production d'oligosaccharides
DE69113028T DE69113028T2 (de) 1990-03-02 1991-03-01 Biochemisches verfahren zur herstellung von oligosacchariden.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9000758-4 1990-03-02
SE9000758A SE9000758L (sv) 1990-03-02 1990-03-02 Biokemiskt foerfarande

Publications (1)

Publication Number Publication Date
WO1991013164A1 true WO1991013164A1 (fr) 1991-09-05

Family

ID=20378752

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1991/000165 WO1991013164A1 (fr) 1990-03-02 1991-03-01 Procede biochimique de production d'oligosaccharides

Country Status (5)

Country Link
EP (1) EP0517766B1 (fr)
DE (1) DE69113028T2 (fr)
ES (1) ES2078512T3 (fr)
SE (1) SE9000758L (fr)
WO (1) WO1991013164A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005936A1 (fr) * 1986-04-02 1987-10-08 Johansson Hanna Maria E Procede de fabrication d'oligosaccharides
US4788145A (en) * 1982-11-11 1988-11-29 Suddeutsch Zucker Aktiengesellschaft Process for preparing 1-O-alpha-D-glucopyranosido-D-fructose
EP0301522A2 (fr) * 1987-07-27 1989-02-01 Showa Denko Kabushiki Kaisha Procédé de préparation d'isomaltulose
US4895801A (en) * 1986-10-07 1990-01-23 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharides
US4957860A (en) * 1986-10-27 1990-09-18 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharide

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE466403B (sv) * 1988-03-24 1992-02-10 Kurt G I Nilsson Saett att syntetisera oligosackarider
SE465516B (sv) * 1989-08-18 1991-09-23 Kurt G I Nilsson Saett att framstaella en oligosackaridfoerening varvid glykosidas fraan en mollusk anvaendes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4788145A (en) * 1982-11-11 1988-11-29 Suddeutsch Zucker Aktiengesellschaft Process for preparing 1-O-alpha-D-glucopyranosido-D-fructose
WO1987005936A1 (fr) * 1986-04-02 1987-10-08 Johansson Hanna Maria E Procede de fabrication d'oligosaccharides
US4895801A (en) * 1986-10-07 1990-01-23 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharides
US4957860A (en) * 1986-10-27 1990-09-18 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharide
EP0301522A2 (fr) * 1987-07-27 1989-02-01 Showa Denko Kabushiki Kaisha Procédé de préparation d'isomaltulose

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN, Vol. 12, No. 326, C525, Abstract of JP 61-237075, publ 1988-04-21 Yakult Honsha Co Ltd. *

Also Published As

Publication number Publication date
SE9000758L (sv) 1991-09-03
DE69113028D1 (de) 1995-10-19
EP0517766A1 (fr) 1992-12-16
EP0517766B1 (fr) 1995-09-13
SE9000758D0 (sv) 1990-03-02
ES2078512T3 (es) 1995-12-16
DE69113028T2 (de) 1996-05-02

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